Project Summary We will develop a method called SPT-RESOLFT that allows the efficient measurement of protein-protein interaction (PPI) dissociation rates and stoichiometry of small complexes in live cells. The approach makes use of Single Particle Tracking (SPT) and Reversible Saturable Optical Fluorescence Transitions (RESOLFT). Fluorescence tags (labeling the proteins of interests) are activated within a small, ~ 100 nm diameter region. The PPI lifetimes and stoichiometry are then inferred from the SPT trajectories as the small number of activated proteins diffuse away from the activation spot. Stoichiometry is determined by change-point analysis that quantifies single-step photobleaching. Dissociation rates are determined by the directly observed divergence of two (or more) trajectories that were previously correlated in motion. In this project, we will specifically target interactions between membrane proteins. This method will speed up the data collection throughput by 10 to 100 times as compared to multi-color SPT methods as well as provide stoichiometry information not available from tracking a sparse subset of proteins as done with traditional SPT methods. Our method will be capable of measuring both homo- and hetero-interactions. Homo-interactions will be measured using a single-color fluorescent protein (FP) approach that takes advantage of the green-emitting photo-switchable FP Dronpa. Hetero-interactions will use a two-color approach that combines Dronpa and the red-emitting photo-activatable protein PAtagRFP. We will verify the method using positive and negative controls with defined labeling stoichiometry as well as with the well-characterized Epidermal Growth Factor Receptor (EGFR, ErbB1) system that changes dimerization kinetics upon stimulation with the EGF ligand. Since dynamic molecular interactions are universally required for responses to signaling inputs, the new tools generated will find application in many different signaling systems. The advantages of higher throughput and increased labeling density will open new possibilities for the study of PPI under a variety of conditions, including ligand dose responses and the influence of therapeutics.